THE ELUSIVE SEARCH FOR FETAL INFECTION

FETAL DIAGNOSIS AND THERAPY 0889-8545/97 $0.00 + .20

THE ELUSIVE SEARCH FOR FETAL INFECTION

Changing the Gold Standards

Carl P. Weiner, MD

Fetal infection due to transplacental passage of virus during maternal viremia is well documented.28, 29, 32* 42, 45, 56 The spectrum of fetal involvement ranges from asymptomatic disease to death or long-term sequelae in survivors depending on the gestation the infection occurs, the virus, and the viral load. Rubella, cytomegalovirus (CMV), herpes simplex virus (HSV), and the varicella-zoster virus (VZV) are known teratogens.13, 37, 38, 56 These agents share the features of ready identification either by culture or by causing a recognizable syndrome in the mother. Congenital malformations are also associated with coxsackie- virus, echovirus (cardiac, urologic, and gastrointestinal malformations), mumps (endocardia1 fibroelastosis), and influenza virus (CNS and heart defect~).'~ Toxoplasmosis and syphilis are well-documented causes of fetal pathology.' Ultrasound findings of intrauterine growth retardation (IUGR), nonimmune hydrops (NIH), isolated ascites, microcephaly, hy- drocephaly, and intracranial or intrahepatic calcification are all consistent with fetal viral infection.*l An infectious etiology is documented by viral culture or serologic methods in 5% to 15% of fetuses with hydrops or isolated ascites, pleural effusions, or pericardial effusions.6, 34 No potential cause is identified in up to 22% of cases of NIH, however, raising the possibility that infection may be even more common than is currently recognized.

Lay and professional journals report two extremes-newly recog-

From the Department of Obstetrics, Gynecology, Reproductive Sciences, and Physiology, University of Maryland School of Medicine, Baltimore, Maryland

OBSTETRICS AND GYNECOLOGY CLINICS OF NORTH AMERICA

VOLUME 24 * NUMBER 1 * MARCH 1997 19

20 WEINER

nized viral infections and new applications of genetically engineered viruses. The recognition that other viruses can cause infection in the adult patient and the application of "harmless" viruses to gene therapy make the elucidation of the full scope of infection during pregnancy essential. There are many standard textbook chapters and review articles on specific perinatal infections; the reader is referred to these for specific questions. The purpose of this article is to provide an update on both the changing methodology and clinical approaches to the diagnosis of fetal infection and the previously unrecognized viruses that may be major pathogens in humans.

TRADITIONAL APPROACH TO THE DIAGNOSIS OF FETAL VIRAL INFECTION

Historically, the identification of a fetal viral illness required recognition of a neonatal syndrome associated with an epidemic of recognizable maternal disease. Rubella is the classic example. The presumed infectious agent then is sought by either culture of the fetus or by the demonstration of IgM antibodies in the fetal circulation. Generally, in the absence of either it is assumed the fetus is uninfected.

This approach requires several crucial assumptions that may be unsustainable. First, it assumes that maternal and fetal infections usually produce a recognizable symptom complex. Second, it assumes that fetal sequelae are present and detectable during the neonatal period and that the fetal immunologic response is sustained until delivery. Third, it assumes that both mother and fetus respond immunologically to a primary infection with the production of IgM antibodies.

PROBLEMS WITH THE TRADITIONAL APPROACH

Maternal viral disease is often asymptomatic, making linkage to a poor pregnancy outcome a problem. The lack of a clinical response by the woman to a virus is not necessarily reassuring. For example, a large group of women were studied during a parvovirus B19 e~idemic.5~ No patient with clinically symptomatic disease experienced a fetal loss. In contrast, women with asymptomatic seroconversion had a 44% loss rate.

The sequelae of fetal infection are often not apparent at birth, providing no opportunity to link their subsequent manifestations with an antenatal event. For example, approximately 90% of CMY-infected newborns are asymptomatic at Fifteen percent of the asymptomatic newborns develop later manifestations, such as sensory neural deaf- ness. More important is whether the fetal infection is the result of a primary infection or a reactivation of the virus during pregnancy.I6 At least 25% of children infected during the primary viremia develop sequelae compared to only 8% of those infected during a reactivation. Furthermore, the sequelae associated with a reactivation are less severe

THE ELUSIVE SEARCH FOR FETAL INFECTION 21

than that associated with a primary infection. Asymptomatic disease (maternal and congenital) presents a similar problem with toxoplasmosis. In one recent report from Massachusetts, 96% of newborns with congenital toxoplasmosis detected by serologic screening were asymptomatic, but more than 40% had some sequelae of infection when examined in greater detail than is typical during the neonatal period.**

Maternal serologic investigation assumes that a maternal serologic response to a primary infection consistently occurs during pregnancy. We have observed several fetuses infected with B19 whose mothers had B19 viral particles in their respiratory excretions and who were serologically negative for B19 until several weeks postpartum, however.@ How often this phenomenon occurs is unknown because serology is typically considered the gold standard for diagnosis.

Even when there is an immunologic response, the maternal serology may be difficult to interpret and requires a convalescent sample 2 to 3 weeks after the acute titer. Both the caregiver and the patient may become unnecessarily concerned and medically confused by this. For example, women with CMV episodically increase their circulating IgM levels in response to reactivation of latent infection.57 Although CMV reactivation poses a risk to the fetus (as noted previously), it is unclear whether a reactivation should be pursued with the same diagnostic vigor as a primary infection. Reactivation is, however, among the most common reasons for referral of a woman with positive CMV serology. Toxoplasmosis serology is also frequently difficult to interpret and in part has led to the FDA's denying permission to the importation of spiramycin to treat presumed maternal toxoplasmosis unless the serologic diagnosis is confirmed by their reference laboratory. A serologic investigation requires a thorough knowledge of the most common infections in the given geographic area.

There is no question that the fetus can respond immunologically to a variety of infectious agents in the absence of a positive culture. The fetal response to infection may be less predictable prior to 20 weeks' gestation, however, based on the observation that 79% of fetuses infected with culture-proven toxoplasmosis are serologically negative for toxoplasmosis-specific IgM in the mid-second trimester." There is also evidence that the fetus may mount an initial immune response that is not sustained. Case reports have documented fetuses who were pathogen- specific IgM positive (e.g., CMV, varicella) early in gestation but IgM negative when born weeks to months later.lO, 23 In the instance of CMV, the infected newborns were either still viruric or had viral particles detectable in their excretions after delivery. Similar responses were observed for parvovirus B19.

Viral culture and identification are generally slow, and certain viruses (eg., parvovirus B19) do not grow readily in standard culture systems.4* The limited amount of specimens available for study limits the diagnostic search to a few likely viruses. A negative culture therefore in no way excludes the possibility of a fetal infection.

The limitations highlighted for both serology and culture suggest

22 WEINER

that the incidence of fetal infection, as assessed by either serologic screening or culture, probably will result in a significant underestimation of the true prevalence of the problem.

NONTRADITIONAL TECHNIQUES

Indirect Fetal Parameters

Infection has a pleomorphic effect on hematologic and biochemical fetal parameters. Anemia, reticulocytosis, polycythemia, hemolysis, thrombocytopenia, lymphocytosis, lymphopenia, and neutropenia are observed in fetuses infected with either viruses or parasites.18, Other indirect evidence of infection includes the development of natural killer cell lymphocytosis. Those fetuses with sonographic manifestations of their disease typically have abnormal transaminase measurements. Viral infection also can cause fetal hypoxemia and acidemia, presumably secondary to either acute or chronic placentitis. In those instances, the umbilical artery Doppler resistance index typically is elevated. It is essential that gestationally appropriate norms for the fetus and not for the neonate or adult be applied. These norms have recently been assem- bled into a single source.%

Electron Microscopy

The application of electron microscopy to the identification of viral particles in the 1980s provided new insights into the occurrence of fetal infection. A variety of techniques made it possible to find and character- ize the morphology of viral particles in fetuses, with indirect evidence of fetal infection but negative pathogen-specific IgM studies and a normal total IgM measurement.@ Either a targeted IgM search or an IgG- specific antibody staining of the viral particles is possible once the family of virus has been identified. Even if all subsequent studies are negative, identification of viral particles in the fetus demonstrates the probable cause of the problem is infection. Although a major advance, electron microscopy is not perfect. The process is time-consuming and not widely available. In addition, the size of a viral load which is necessary for detection is unknown.

PCR

Polymerase chain reaction (PCR)39, 53 allows rapid detection of specific viral nucleic acid sequences in infected tissues and body fluids from adults, children, and fetuses.2, 9, 50, 51, 54, 67 The detection of a fetal infection with CMV, toxoplasmosis, adenovirus, and human parvovirus B19 by PCR has been reported in case reports and small series.24, 52,


One investigator concluded that nested PCR was the method of choice for the diagnosis of congenital CMV in women referred for positive CMV serology.25 PCR also has been applied to the identification of viruses in adults and children with myocarditis and dilated cardiomyop-

In collaboration with Towbin, this author recently used PCR to determine the association between viral genomic material and sonographically abnormal pregnan~ies.~~ The oligonucleotide primers used were designed to amplify nucleic acid sequences from seven common viruses known to cause fetal infection, to cause myocarditis in infants and children, or to infect persons with marginal immune system func-

This routine application of PCR yielded startling results, the impli- cations of which will take years to understand. Viral genomic material was identified in over 40% of sonographically abnormal pregnancies (Table 1). The abnormalities involved were diverse (e.g., fetal growth restriction, nonimmune hydrops, "stuck-twin" syndrome, oligohydramnios, hydramnios, ventriculomegaly, echogenic bowel, and structural malformations). In contrast, the incidence of positive studies in women undergoing genetic amniocentesis for advanced maternal age with a normal sonogram was fewer than 2.5%.

Surprisingly, adenovirus was the most commonly detected virus, occurring twice as often as CMV (24% vs. 10%; Table 2). Adenovirus previously had only rarely been implicated in fetal disease. The enterovi-

athy.19, 20. 26, 35, 66

tiOn.7, 14,31,41, 44,49

Table 1. INDICATION FOR SAMPLING VERSUS PCR RESULT

Indication Patient PCR + PCR -

Number (n) (%I (%I ~ ~~

Nonimmune hydrops Oligohydramnios Hydramnios HydrothoraxJpleural effusion Stuck-twin syndrome Ventriculomegaly Microcephaly Cystic hygroma Choroid plexus cyst Echogenic bowel Liver calcification IUGR Thick placenta Hydronephrosis Multiple congenital anomalies Myocarditis (autopsy-proven) Maternal infection

91 1 1 15 18 25 26 5 7 5 22 3 22 7 3 7 12 22

~

41 (45) 7 (64) 8 (53)

19 (73) 4 (80) 7 (100) 5 (100)

2 (66) 12 (54) 7 (100) 3 (100) 7 (100) 2 (17) 10 (45)

12 (66) 15 (60)

16 (73)

Total Normal Ultrasound

301 124 (41) 179 (59) 154 4 (3) 150 (97)

Modified from Van den Veyver 16, Ni J, Carpenter RJ: Detection of intrauterine viral infection using the polymerase chain reaction (PCR). American J Obstet Gynecol, in press, with permission.

Tabl

e 2.

VIR

AL

ETI

OLO

GIE

S OF

FETA

L D

ISE

AS

E

Vira

l Gen

ome

Am

plifi

ed (n

) PC

R +

Indi

catio

ns (N

o. o

f P

atie

nts)

P

atie

nt N

o.

Ade

no

Ent

ero

Par

vo

CM

V H

SV

EBV

RSV

To

tal

Non

imm

une

hydr

ops

(91)

O

ligoh

ydra

mni

os (1

1)

Hyd

ram

nios

(15)

H

ydro

thor

ax/p

leur

al ef

fusi

on (1

8)

Stu

ck-tw

in sy

ndro

me

(25)

V

entri

culo

meg

aly

(26)

M

icro

ceph

aly (

5)

Ech

ogen

ic b

owel

(22)

Li

ver c

alci

ficat

ion

(3)

IUG

R (2

2)

Myo

card

itis

(aut

opsy

-pro

ven)

(1 2)

M

ater

nal i

nfec

tion

(22)

50 4 7 6 10 7 1 6 1 10

10

12

30'

2 5*

4 4*

6 1 1 0 8*

6*

7*

7* 0 3*

1 5*

0 0 0 0 1' 5*

0

8*

0 0 0 0 0 0 0 0 0 0 0

8*

2 0 1 2"

1 0 5' 1 3' 1*

6'

5*

0 0 0 2*

0 0 1' 0 0 0 1"

2 0 0 0 2*

0 0 0 0 0 0 0

1'

0 0 0 0 0 0 0 0 0 0 1*

61* 4 8*

6 1V

7 1 7*

1 12'

12*

157

Tota

l (30

1)

Nor

mal

Ultr

asou

nd (1

54)

124

74

22

8 30

9

4 2

149

4 3

0 0

1 0

0 0

4

Mod

ified

fro

m V

an d

en V

eyve

r IB, N

i J, C

arpe

nter

RJ:

Det

ectio

n of

int

raut

erin

e vi

ral i

nfec

tion

usin

g th

e po

lym

eras

e ch

ain

reac

tion

(PC

R).

Am

eric

an J

Obs

tet G

ynec

ol, i

n pr

ess,

with

per

mis

sion

.


ral genome was amplified from 7”/0 of the patients in our study. Perhaps this should not be surprising because enteroviruses, especially coxsackie- virus, are common causes of illnesses such as myocarditis in neonates and older ~hildren.~, 6, 19, 26 Enteroviral infections have been reported only rarely as a cause of hydrops, however. There are reports of newborns with enteroviral myocarditis diagnosed so early in life that the infection must have been contracted during the peripartal period, h~wever .~, 27, 30

Adenoviruses, and less frequently CMV and HSV, also have been reported to cause myo~ard i t i s .~ ,~ ,~~ , 58 In the experience of this author, there have been only two cases of prenatally acquired adenoviral myocarditis reported? one of which was diagnosed using PCR. This fetus was successfully treated in utero with cardiac supportive therapy (digoxin),62 as was an earlier case of myocarditis secondary to parvovirus.M More recently, adenovirus was shown to produce inflammation in the sheep

These findings cast doubt on the wisdom of using adenovirus as a vector for fetal gene therapy.

Certainly questions remain. Does the presence of viral genomic material in amniotic fluid, fetal blood, or fetal effusions represent fetal infection that has caused the pathology, or is the “sick” fetus more susceptible to transplacental infection? Both are possibilities. If we exam- ine our control group in detail, we see that the PCR studies were positive in 8% (13 of 163). Nine fetuses from the control group had to be recategorized because they subsequently developed sonographic abnormalities (69%); (NIH, ventriculomegaly); three of these fetuses died. This high complication rate for apparently normal but PCR-positive pregnancies at the time of genetic amniocentesis suggests that a positive PCR indicates fetal infection, and it also suggests that the presence of viral genomic material at the time of second-trimester amniocentesis is a major risk factor for an adverse pregnancy outcome. Positive PCR accounted for all late losses (> 7 days) after genetic amniocentesis in our center at the University of Iowa (Unpublished data, 1996). The secondary observation, that fetuses with genomic material from more than one virus had a higher perinatal mortality rate, is consistent with increased susceptibility. CMV was typically found to be associated with other viruses. Because of the large number of centers involved and the incomplete follow-up, we cannot yet determine whether a positive PCR increases the risk of a perinatal death for any given sonographic abnormality. Studies are underway at our two institutions to address this issue.

Could the difference in prevalence of positive PCR studies in sonographically normal and abnormal pregnancies reflect the 10-week difference in the gestational ages at testing? Whereas it is likely the prevalence of positive PCR studies will rise with advancing gestation, it seems unlikely to increase 20-fold. Studies at our institution are currently testing specimens obtained from normal near-term pregnancies to address this issue.

Could the positive PCR studies represent maternal contamination occurring as the needle passed through the maternal abdomen? Al-

26 WEINER

though the possibility of such an event cannot be ruled out, it seems unlikely that contamination occurred 20 times more often in the group with an abnormal ultrasound evaluation.

Although the detection of viral genome in 41% of our study population is considerably higher than was previously reported? 49 the earlier studies may have been compromised by the low sensitivity of standard viral culture methods and serology for many of the viruses studied. Positive diagnosis of fetal infection will likely move from serology and culture to PCR within the next 5 years.

RECOMMENDED APPROACH TO THE SEARCH FOR FETAL INFECTION

The approach to search for fetal infection depends on how the patient presents for evaluation. These are the approaches presently used at The Maryland Center for Advanced Fetal Care.

Abnormal Maternal Serologic Screen

The utility of these screening tests recently has been reviewed.15 Their value reflects the likelihood of a primary infection during pregnancy, the accuracy of the test (and the likelihood of a maternal serologic response), the likelihood of fetal infection (either vertical or horizontal), the accuracy of fetal diagnosis, the fetal sequelae, and the potential for prevention or treatment.

Hepatitis and HIV are not known to be associated with a fetal syndrome. Screening for Toxoplusrna becomes cost effective when the maternal infection rate exceeds 1 in 1000.59 This rate excludes virtually all of the United States. Screening for syphilis is cost effective when the maternal infection rate exceeds 5 in 100,000.15 Many states, however, mandate serologic screening for syphilis even in low-risk populations. The prevalence of congenital rubella in countries with universal vaccina- tion is now so low that it precludes cost efficacy, but the sequelae are so great should it occur in the first trimester that most authorities recommend routine screening of pregnant women.

Screening for CMV, a common practice in parts of the United States, is problematic. Specific IgM antibodies are found in 80% of women with primary CMV infection and may last as long as 7 months. CMV-specific IgM antibodies reappear in about 20% of women with a CMV reactivation. Primary infection can be diagnosed with certainty onZy if either there is a documented seroconversion during pregnancy or if the IgG is negative and IgM positive when the subject is first tested with a subsequent seroconversion in IgG. Detection of primary infection is difficult and creates needless anxiety in many women who have experienced a reactivation. Counseling the women with either seroconversion or reactivation but a normal ultrasound examination cannot be based on


hard data (see discussion that follows), nor have effective therapeutic interventions for the treatment of fetal CMV infection been identified.47 We believe that routine screening for CMV is not medically justified.

A positive serologic test for a virus or parasite associated with congenital disease requires additional testing. The very high association of first-trimester rubella with malformations justifies the option of pregnancy termination in the absence of any additional work-up. The diagnosis of a primary Toxoplasma infection, because of testing vagaries, requires confirmation by a reference laboratory if this is not done initially.

Abnormal screening test results should be followed with a targeted ultrasound examination. The RADIUS Trial demonstrates that this examination should be performed by someone with considerable experience evaluating abnormal pregnancies. Although the presence of a sonographic abnormality and a positive screening test strongly suggests the fetus is infected, a normal sonographic examination does not rule out pathology. A sonographically normal fetus may be either uninfected, infected but unaffected by the disease process, or infected and affected by the disease process but in a manner not sonographically detectable. This uncertainty makes counseling difficult and options ranging from pregnancy termination to no action should be carefully examined with the patient and her partner (Fig. 1). The correct approach to pregnancy management is what the patient is most comfortable with doing. She needs to be informed that a normal scan is reassuring but does not exclude the possibility of fetal infection.

Symptomatic Maternal Illness

Symptomatic maternal viral disease typically indicates primary infection and thus carries a greater risk for the fetus. The physician response to a symptomatic maternal illness by an unidentified agent is similar to the sonographic follow-up of a positive serologic screen. Although the initial PCR work suggests that many common viral infections in adults do pose a risk to the fetus, it is too early in the experience of this author to recommend a search for these agents routinely. Their implication when associated with a normal ultrasound evaluation is not yet clear. These tests are undertaken in the Maryland Center for Ad- vanced Fetal Care as a part of ongoing research.

Abnormal Ultrasound Evaluation

The PCR studies cited indicate that virtually any sonographic abnormality may be the result of a fetal viral infection. They further illustrate that the pathology continues to evolve. A normal scan at 18 weeks does not preclude the possibility of a markedly abnormal scan later in gestation. Because the fetal biochemical and serologic response to infection may be transient, failure to evaluate an abnormal pregnancy when

28 WEINER

Counseling re: Potential for Dx Risk of Dx Predictive value of a +Dx

reassurance)

I divine

confidence

(Treat] (-1 [-] Termination

Figure 1. Algorithm of maternal illness or + serology in pregnant women. Dx = Diagnosis.

it presents because of a lack of treatment options potentially wastes the opportunity to make a diagnosis in the future.

The argument that there is no need to diagnose a viral disorder when we cannot yet change outcome is shortsighted. First, the fetus or newborn may die either directly secondary to the infection or indirectly from preterm delivery. Should that occur, the patient and physician are left not knowing the etiology, and the physician is unable to counsel the patient on the risk of recurrence. Second, knowledge that a growth- restricted fetus is small because of infection rather than hypoxemia will prevent many iatrogenic premature births having been based on the faulty assumption that the fetus is small because of hypoxia. The same may be said of the fetus with ascites secondary to chronic hepatitis. In these cases, early delivery in the face of normal cardiac function is unlikely to improve outcome. Third, many congenitally infected fetuses continue to shed virus throughout the neonatal period. These newborns should be isolated from reproductive-aged women who might be at risk for infection. Fourth, continued progress is being made on the development of antiviral agents. It may well be possible to treat these fetuses in the future and prevent further progression of the abnormalities. The Maryland Center for Advanced Fetal Care, in collaboration


with the Maryland Institute for Human Virology, has a major interest in such therapies.

CONCLUSION

Two percent to four percent of all newborns have a major structural malformation of which only a small percentage can be explained by either aneuploidy or a single-gene disorder. No other mammal has a similar reproductive problem. It is the suggestion of this author that previously unrecognized viral infection accounts for a large percentage of these abnormalities by interfering with appropriate embryonic cell migration, reducing hyperplasia, or damaging the precursor structure by means of an inflammatory mechanism.

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Carl P. Weiner, MD Department of Obstetrics, Gynecology, and Reproductive Sciences

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THE ELUSIVE SEARCH FOR FETAL INFECTION